LT3 NMR Side-Chain Assignment Flashcards
What don’t we know after doing HNCACB and CBCAA(CO)NH?
We know the correct order of the residues
But don’t know which amino acid the peak is yet
What does TOCSY stand for
Total Correlation Spectroscopy
For ALIPHATIC side chain assignment
What is TOCSY used for?
Correlates amide groups (N-15 labelled) to all aliphatic H (protons) in the SAME amino acid residue
What will each amide strip of a TOCSY show?
Each amide strop will show H chemical shift of Ha, Hb, Hy from the same residue in the 3D
In a TOCSY, can we always see all aliphatic protons?
No
If transfer is not enough, sometimes only Ha and Hb can be observed
What are the axes of the TOCSY?
H1 vs NH
How do we get secondary structure from chemical shift index?
Secondary shift = different between H/C chemical shift for each residue and that reported for the same residue type in a “random coil” conformation
Experimental - random coil = secondary
What does the Chemical Shift Index (CSI) tell us?
Correlates between chemical shift tendencies and secondary structure
What are the values of a Ha or Cb secondary shift?
-ve for alpha helix
+ve for beta-sheet
What are the values of a Ca secondary shift?
+ve for alpha helix
-ve for beta-sheet
What does NOE stand for and what is it?
Nuclear Overhauser Effect = exist only between two H separated by less than 5 angstrom
What does an observable NOE tell us?
Means the two H are in close vicinity to each other
Describe the difference in NOE for the secondary structures
Alpha-helix = characterized by the close approach between residue i and i+3
Extended beta-sheet = excludes medium range distances
Close proximity of 2 strands brings about inter-strand distances
What is the difference between N-15 TOCSY and NOESY?
TOCSy will only protons on that SAME residue
NOESY will only show protons within 5 angstrom = including side chains from other residues
What does intensity on a NOESY tell us?
How close the H are to each other
What is NOE cross-peak intensity proportional to?
1/r^6
Why do HSQC-TOCSY and HSQC-NOESY show different things?
TOCSy = through bond expt, gives chemical shift of all H within same residues as the HN
NOESY = through space expt, gives chemical shift of all H within a distance of 5A from particular HN (amide proton)
What do the different dxy(i, i+z), dNN(i,i+1), daN, dbN show?***
Define intra-residue NOE
Between H within the same residue
Name the 3 inter-residue NOE
Sequential = adjacent residues
Medium range = i to i+4
Long range = more than 4 residues apart
Definer inter-molecular NOE
Between two molecules = dimer or beta-strands
What NOEs define what structures of proteins?
Secondary = short and medium range
Tertiary = long range
Quaternary = inter-molecular
Are daN and dNa different in NOE?
YES
Depict different places between residues
What NOEs can we find in alpha-helix?
Sequential and medium range
dNN(i, i+1), daN(i, i+3), daN (i, i+4) and dbN(i, i+1)
What NOEs can we find in beta-sheet?
Sequential and inter-strand
daN (i, i+1)
What type of NOE defines tertiary structures?
Long-range NOEs
How can we tell if beta-sheets are parallel or anti-parallel?
Parallel = N and N terminals will be close and C-C terminals will be close
Antiparallel = N-terminus close to C-terminus
Are anti- or parallel beta-sheets closer and why?
Antiparallel sheets have closer beta-strands because the H bonds are more linear
What is the coupling constant between?
J = measured between pairs of protons separated by 3 covalent bonds
Contains information about intervening torsion angle
Which dihedral angle is used for J and why?
Phi angle
Because related to both secondary and tertiary structure
What is the coupling constant for alpha-helices and beta-sheets?
a-helices = 4Hz
b-sheets = 9Hz
What are the phi angles for right-handed a-helix and anti-parallel b-sheet?
a-helix = -57
b-sheet = -139
Larger angle = larger distance in Hz of the peak
What are labile protons, and what happens to them in the presence of heavy water (D2O)?
Labile protons = easily exchangeable (–NH or -OH)
In presence of heavy water, H-1 get exchanged for H-2
H-2 cannot be detected by HSQC because gives out different resonance freq
How are hydrogen bonds useful in determining standard secondary structure and tertiary structures in NMR?***
What 3 restraints are usually used for NMR structure calculation?
NOE distance restraints=strong, medium, weak
Dihedral angle restraints from coupling constant
Hydrogen bond restraints
What does the computer do to generate an ensemble of structures that obey the constraints?
Use simulated annealing and energy minimization
What happens if the number of constraints is larger?
The ensemble structure will be very similar to each other
Having a SMALL root mean square deviation (rmsd) between their backbone and side chain
Summarize the steps of protein NMR
Get a solution of proteins with C-13 and N-15
Triple resonance 3D experiments
Backbone and side chain sequential assignment by through-bond experiments
Distance and dihedral angle restraints through-space and coupling constant experiments
What are the cons to NMR?
Protein size is limited
High solubility is needed
Conformational exchange problems
Distance restraints assignment and structure refinement
What are the prerequisites needed for NMR?
Over-expression of folded protein in minimal medium
~95% purity
Highly soluble
Stable at greater than or equal to 25 degrees for a week
